DESCRIPTION: Diabetic foot ulcers (DFU) comprise a large part of chronic wounds with an estimated annual incidence of more than 750,000 new cases per year. It is currently not known whether differences exist in wound healing processes been T1DM and T2DM, although they are assumed to be the same. The hypothesis underlying this proposal is that the persistence of M1 polarized macrophages actively inhibit healing of diabetic wounds, and that redirection of the inflammatory response to the pro-regenerative M2 phenotype will enhance healing of these wounds. We also hypothesize that T1DM patients who are prone to develop DFU have increased pro-inflammatory state that is similar to T2DM patients. In order to test our hypothesis, we will first test how M1 and M2 macrophages impact wound healing in 3D, in vitro human skin-like tissues, and how the diabetic tissue microenvironment modulates the polarization of macrophages. To this end, we will utilize 3D, skin-like tissues composed of cells derived from streptozotocin (STZ)-induced T1DM (STZ-DM), db/db T2DM and non-diabetic subjects to mimic chronic and normal wounds, and to: a) determine if macrophages pre-polarized to the M2 phenotype, as versus M1, enhance healing of damaged tissues; b) study if tissues engineered from diabetic cells direct the acquisition of the M1 phenotype from nave cells; c) learn how functional wound responses are accelerated or inhibited by M1 and M2 macrophages by dissecting paracrine signals directing normal and chronic healing; d) identify possible differences between T1DM and T2DM. We will then confirm the ability of monocytes polarized to M1 and M2 in vitro, to impact healing of neuropathic wounds in diabetic animals when introduced to the wound site, and determine the propensity of nave monocytes derived from T1DM and T2DM to polarize to the M1 phenotype at the wound site. For this, wounds will be created in STZ-T1DM and db/db (T2DM) and non-DM mice, and serum monocytes either in a nave state or first be polarized to the M1 and M2 phenotype in vitro will be applied. Wound healing progression, macrophage polarization and possible differences between T1DM and T2DM will be assessed. Finally, we will test the ability of biomaterials that sequentially release factors to first recruit and then direct the M2 phenotype to enhance healing in diabetic, neuropathic rodent wounds. We will develop wound dressing materials that provide a localized and sequential release of first MCP1 or Substance P to recruit monocytes, and then IL-4 or IL-10 to direct the M2 phenotype, and test their wound healing capacity in the T1DM and T2DM mice and diabetic neuroischemic rabbit ear wound model. Successful completion of these aims will allow us to determine the impact of macrophage phenotype on wound healing, dissect differences between T1DM and T2DM and design new biomaterials capable of modulating the local inflammatory response and modulating wound healing. Altogether, these results may lead directly to a new strategy, and new therapies, to treat chronic, diabetic ulcers in T1DM and T2DM patients.